Nanodrugs based on peptide-modulated self-assembly: Design, delivery and tumor therapy

Accepted Manuscript Nanodrugs based on peptide-modulated self-assembly: Design, delivery and tumor therapy

Shukun Li, Ruirui Xing, Rui Chang, Qianli Zou, Xuehai Yan PII: DOI: Reference:

S1359-0294(17)30124-3 https://doi.org/10.1016/j.cocis.2017.12.004 COCIS 1155

To appear in: Received date: Revised date: Accepted date:

8 October 2017 13 December 2017 13 December 2017

Please cite this article as: Shukun Li, Ruirui Xing, Rui Chang, Qianli Zou, Xuehai Yan , Nanodrugs based on peptide-modulated self-assembly: Design, delivery and tumor therapy. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cocis(2017), https://doi.org/10.1016/ j.cocis.2017.12.004

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ACCEPTED MANUSCRIPT Nanodrugs based on peptide-modulated self-assembly: design, delivery and tumor therapy Shukun Li1,3, Ruirui Xing1, Rui Chang1,3, Qianli Zou1,2, and Xuehai Yan1,2,3*

State Key Laboratory of Biochemical Engineering and 2Center for Mesoscience, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 North Second Street, Zhongguancun, Beijing 100190, China 3

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1

University of Chinese Academy of Sciences, Beijing 100049, China

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Web: http://www.yan-assembly.org

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Email: [email protected]

ACCEPTED MANUSCRIPT Abstract: In this review we consider assembled nanodrugs as a type of nanoscale drugs formed by molecular self-assembly and associated with precise organization of multiple non-covalent interactions. Their typical feature is that the drug itself is considered as one of the building blocks with flexibly interplaying interaction for supramolecular

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assembly and nanostructure formation with robust stability and high loading efficiency in a controlled and tunable way. The super stability with retained function

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results from the “hydrophobic effect” of supramolecular self-assembly of peptides and

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drugs. It is the hydrophobic effect responsible for both colloidal stability and circulation stability in body against dilution and blood-flow shearing. The assembled

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nanodrugs are distinguished from conventional ones with encapsulation of the drugs in delivery nanocarriers. We will focus on how peptides and peptide-conjugates can

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be designed for controlling and mediating the formation of the assembled nanodrugs. Emphasis will be put on the rational design of intermolecular interactions between

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drugs and peptides, in vitro and in vivo drug delivery and antitumor therapeutic

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effects. Finally, we will discuss the key challenges and promising perspectives of such kind of peptide-mediated assembled nanodrugs for both technical advances and

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potential clinical translation.

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Keywords: peptides; intermolecular interactions; self-assembly; nanodrugs; tumor therapy

ACCEPTED MANUSCRIPT 1. Introduction The convergence of medicine and nanotechnology has led to the field of formation of nanodrugs exploiting supramolecular materials to optimize targeting, desired local pharmacokinetics and minimize systemic toxicity over the course of treatment [1-9]. Encouragingly, all research efforts are being increasingly translated to clinical

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applications [10, 11]. Nature guides rational ways of supramolecular materials construction, as exemplified by protein folding, DNA double-helix and phospholipid

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bilayer membrane formation, in harnessing the power of molecular self-assembly.

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Self-assembly is a “bottom up” fabrication method to organize molecular components into well-ordered architectures via non-covalent interactions spontaneously under near

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thermodynamic equilibrium conditions. Non-covalent interactions as electrostatic forces, van der Waals forces, π interactions (π-π stacking, cation-π interactions and

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anion-π interactions) and hydrophobic interactions [12, 13] drive molecular recognition motifs to form 0 dimensional (0D), 1D, 2D and 3D structures with

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specific, directional, tunable properties in a cooperative way from nanoscale to

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macroscale [14-17].

Over decades of extensive efforts, the accumulated knowledge has successfully

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enabled the creation of a large arsenal of supramolecular structures from an ever expanding set of natural and synthetic building blocks, including liposomes [18, 19],

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polymers [20-23], peptides [24-29], and inorganic nanomaterials based on silica [30], gold [31] and carbon [32, 33]. Simultaneously, the application of these materials has been ubiquitous in the practice of medicine, particularly benefits nanodrugs delivery towards tumor sites [34-37], to a large extent due to the enhanced penetration and retention in tumor lesion as increased vascular permeability and poor lymphatic drainage [38]. As for assembly building blocks taken into consideration, tremendous attention has been and will be more paid on amino acids, peptides, proteins and their derivatives inspired by naturally occurring fabrication motifs. These biomolecules regulated self-assembled nanostructures remain the most attractive option for several

ACCEPTED MANUSCRIPT reasons [25, 39-41]: (i) The inherent biological origin endows the assembled structure with excellent biocompatibility and biodegradability, high tissue permeability and non-immunogenicity thereby guaranteeing pharmacological safety; (ii) The explicit molecular structure allows synthetic feasibility and modification by introducing functional groups, which yield resultant nanostructure with specific features on

individually unattainable.

As

a

whole,

peptide

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demand; (iii) The adopted collective forms possess novel properties that are modulated

supramolecular

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nanostructures promoted the solubilization, controlled release of small-molecule

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pharmaceutics, bioactive proteins or other therapeutically relevant payloads [42-47] with specific applications in anti-tumor therapy. However, in the practical blood

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circulation of nanodrugs, Fickian diffusion governs the drug leakage randomly, tumor interstitial fluid pressure limit [48] and the stochastic nature of ligand-receptor

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interactions provide difficulties to control drug release precisely. This overall represents less specific targeting to cells, tissues or organs. Therefore, a daunting task

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of smarter designs with on-demand stimuli-responsiveness gained increasing attention

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to promote transformation of nanodrugs from “bench to bedside” [49-52]. Active targeting involved in leveraging molecular recognition through natural or engineered receptor-ligand motifs exhibit structural and functional biomimicry [53, 54]. Passive

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targeting enhancement resulting from endogenous stimuli related to a lesion microenvironment [55] may be even used for a hierarchical passive targeting strategy

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[56]. Hence, improved bioavailability facilitates a higher therapeutic index ultimately.

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Fig. 1. Schematic illustration of constructing assembled nanodrugs, the interactions for structure formation, and

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applications towards antitumor therapy.

Notably, extensive efforts have been focused on drug carrier construction. The

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carrier-drug relationship is based on a separation of function, which means the nanocarrier plays a sole role in delivery responsible for maintaining the bioactivity of

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drugs. Once a delivery function fulfilled, the carrier is destined to degrade or be excreted. Sometimes, encapsulation is challenged with leakage issues, lower drug

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loading, batch-to-batch variations and undesired synthetic carrier elimination rate.

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Interestingly, an emerging approach of co-assembly of peptides with drugs has been verified as an effective way to avoid the aforementioned issues [57-59]. The principle

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is similar to DNA base pairing, co-assembly permits the binary components involved in the final structure to participate in the interaction spontaneously via corresponding

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amino acid, peptide, drug and in interactional pairing completely [13, 60-65] or triggered by a helper substance [66, 67]. Co-assemblies possess suitable stability, higher loading efficiency, fully interpenetrated interaction of both components in a controlled and tunable way. Alternatively, designed or synthesized drug-conjugate self-assembly is another way to gain access to, and control over, rather complex nanostructures [68-72]. Comprehensively, both ingenious designs optimize the drug or drug conjugate dual function of intrinsic bioactivity and the molecular building regulation. In this review, we will put emphasis on design of assembled nanodrugs,

ACCEPTED MANUSCRIPT flexible regulation of non-covalent interactions and delivery to tumor sites and therapeutic effects (Figure 1). 2. Peptide-modulated formation of assembled nanodrugs Oligopeptides are recognized as extremely useful building blocks for creating self-assembling nanostructures for medical applications due to their inherent

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biocompatibility and biodegradability. Sometimes modification to bestow the peptide

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with amphiphilicity provides features as amphiphilic surfactants with bioactive

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functions, which are known to assemble into a variety of nanostructures.

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Fig. 2. (a) Schematic depiction of amphiphilic dipeptide- or amino-acid-tuned self-assembly for fabrication of photosensitive nanoparticles. (b) Fluorescence images of the MCF7-tumor-bearing mouse model showing the

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biodistribution of FCNPs and free Ce6. (c) Measurement of tumor growth for 20 days.

Diphenylalanine peptide (FF) is the core recognition motif for molecular self-assembly, which is extracted from β-amyloid polypeptide related to Alzheimer disease and has been observed to form one dimensional nanostructures through the combination of hydrogen bonding and π-π interactions [73-75]. A common modification involving the hybridization with alkyl or aromatic groups gives rise to an additional directional driving force for self-assembly through hydrophobic interactions and/or π-π conjugation [76-78]. Recently, our group [57] suggested, that the FF derivative, cationic diphenylalanine (H-Phe-Phe-NH2·HCl, CDP) acts as an

ACCEPTED MANUSCRIPT amphiphilic model peptide to regulate hydrophobic photosensitizer-Chlorin e6 (Ce6) co-assembly to enhance PDT efficacy. Amphiphilic CDP possesses a phenyl ring providing a hydrophobic moiety and amine groups providing a hydrophilic moiety. Ce6 is a negatively charged photosensitizer and possesses a highly hydrophobic porphyrin ring. The non-covalent interactions including hydrophobic interaction and

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π-stacking interactions, along with electrostatic attraction between positive CDP and negative Ce6, can induce binary components to form nanoparticles with high loading

the

colloidal

nanoparticles

in

the

body

circulation.

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of

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efficiency (Figure 2a). Notably, strong hydrophobic interactions maintain the integrity

9-fluorenylmethoxycarbonyl-L-lysine (Fmoc-L-Lys) was also applied as an

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amphiphilic amino acid to fabricate self-assembled nanostructures, thus proving the universality of regulation. The obtained Fmoc-L-Lys/Ce6 (FCNPs) and CDP/Ce6

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(CCNPs) showed narrow size distribution and a hydrodynamic diameter of 200 nm and 100 nm, respectively, which played a significant role in limiting the further

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aggregation of Ce6 in the eventual structure. Furthermore, the nanoparticles were and enzymes corresponding to the tumor

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responsive to pH, detergents

microenvironment, exhibiting an on-demand release. In vivo biodistribution showed stronger fluorescence of NPs-treated mice at the same time intervals than free Ce6,

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ascribed to the EPR effect of NPs (Figure 2b). In vivo PDT therapeutic test verified that tumors of NPs-treated mice were efficiently suppressed and completely

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eradicated, while the tumor treated with free Ce6 showed rapid recurrence (Figure 2c). Monitoring the body change showed no decrease in the NP-treated group, demonstrating the high biocompatibility of NPs (Figure 2d). Occasionally, a stabilization procedure is necessary to consolidate the structural stability of the supramolecular nanostructures. Various crosslinks can be incorporated into FF derivatives to enhance the physical and chemical robustness. For instance, precross-linking FF derivatives with glutaraldehyde [79] or natural alginate dialdehyde [80] followed by the hydrophobic interaction between water-insoluble

ACCEPTED MANUSCRIPT drugs may attain a relatively stable nanodrug formulation. Somewhat surprisingly, drug can act as cross-linker to assist its association with peptides. The inherent β-sheet-like hydrogen bonding combined with π-π interactions of FF derivatives leveraged to create peptide intermolecular stacking motifs that form filamentous one-dimensional assemblies with the assist of hydrogen bonding between

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molecules parallel to the long axis of these assemblies. Yang and co-workers [81] designed a novel peptide, Nap-GFFYG-RGD solely to form nanofilaments due to the

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relatively weak inter-fiber interactions, enabling encapsulation of curcumin. Ding and

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coworkers [82] co-assembled the peptide Nap-GFFYG-RGD with doxorubicin (DOX), surprisingly finding gel formation due to the suitable interaction assisted by DOX as a

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cross-linker. They found that DOX formed large nanospheres (∼150 nm in diameter) on the fiber surface, due to the electrostatic interactions between the negatively

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charged nanofibers and the positively charged DOX. To some extent, the combination of hydrophobic regions enhances the interaction between peptide nanofibers and DOX

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nanospheres. Nanospheres essentially act as cross-linkers increasing the inter-fiber

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interactions, thus enabling gel formation. DOX drug release was observed to be faster from mechanically weaker gels correlating with the lower concentration of DOX. In vitro test showed that DOX-peptide hydrogels and DOX possess similar anti-tumor

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activity, while the complex may be capable of releasing the drug in a sustained manner, minimizing the drug adverse effects. It is worth mentioning that both works

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[81, 82] conjugated the tripeptide Arg-Gly-Asp (RGD) to the FF derivatives. This enhanced the mediated cell interaction via recognition binding to integrin, as integrin overexpressed in numerous solid tumors and served as a marker of tumor angiogenesis, development, and metastasis. Furthermore, from the structural engineering viewpoint, decoration with structural adjustment groups[83, 84], like ferrocene (Fc) and the most investigated fluorenylmethoxycarbonyl (Fmoc) group, whose aromatic ring can further enhance hydrophobic interaction and π-π stacking and thus promote the formation of supramolecular architectures[85], is also an

ACCEPTED MANUSCRIPT alternative approach to protect N-terminus of FF via capping. In the former case of Fc-FFRGD [83], which provides driving forces via hydrophobic and π-π interactions and thus represents a novel supramolecular hydrogelator to form a high-aspect-ratio nanostructures thermodynamics favored after a morphological transformation. HeLa cells overexpressed integrin αvβ3 on cellular membrane were chose to be model for

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verifying the specific targeting cellar uptake, explained as multivalent RGD-integrin binding enhancement effect. The later instance of Fmoc-FF, conjugation with histidine,

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arginine or cysteine at their C-terminus to provide versatile binding sites for AuNPs

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via coordination, electrostatic interaction and covalent bonding, respectively. As a whole, the design effectively reversed the agglomeration of AuNPs and realized

nanofibers untouched in the process.

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activity optimization. Significantly, the morphology and secondary structure of the

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Exquisite peptide molecular design matching to the corresponding drug molecular may complement non-covalent force to driven co-assembly. Zhang and co-workers

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[61] exploited the complementary hydrogen bonding interaction to trigger

with

hydrogen

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co-assembly of amphiphilic peptide and antitumor drug. Cyanuric acid and melamine bonded

pair

were

conjugated

to

the

hydrophobic

tail

(CA-C11-GGGRGDS) bestowing the designed molecule with amphiphilicity to form

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spherical micelles in aqueous solution. The antitumor drug, methotrexate (MTX), has a structure similar to melamine, which shows great potential to form complementary

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hydrogen bonding interaction with cyanuric acid. Mixing the binary components, hydrogen bonding between MTX and the terminal cyanuric acid group could form complementary hydrogen bonds to improve the overall hydrophobicity, resulting in re-assembling for formation of MTX loaded nanorods in low MTX concentration. Increasing the concentration of the amphiphilic peptide/MTX complex yielded higher hydrophobic aggregation degree and thus induced the formation of nanofibers, in which the assembly morphology transition from spherical micelles to nanofibers is reversible dependent on MTX concentration. Interestingly, when increasing or

ACCEPTED MANUSCRIPT decreasing external pH, a transition of MTX from hydrophobic to hydrophilic would appear and further disturb the balance of the system to increase the release rate, which may be applied as a stimuli-response to exert the following therapeutic effect in the acidic tumor microenvironment. In addition, a variety of functional peptide motifs have attracted increasing

For

example,

Guo’s

group

[87]

assembled

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attention for use as building blocks for fabrication multi-functional assemblies [86-89]. the

bifunctional

peptide

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HAVRNGRRGDGGAVPIAQK (HRK-19) and doxorubicin to obtain DOC/peptide

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NPs. Bifunction referred to the amino acid motifs in the HRK-19 design with specific targeting. Simply, fusing RGD/NGR peptide enhanced tumor targeting and peptide

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sequence-AVPIAQK induced tumor cells apoptosis. Kataoka and co-workers [89] have constructed in vivo nanoreactors based on the co-assembly method, namely

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PICsomes. Negatively charged PEG-b-Asp and positively charged Homo-P (Asp-AP) co-assembly into PICsome via electrostatic interaction. Mechanical stress, whereby

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PICsomes underwent fragmentation into pairs of single aniomer and catiomer

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polymer chains, or unit PICs (uPICs), and reassociation took place after the removal of such perturbations. Because of the reversible association/dissociation, negatively charged enzymes can be loaded particularly. The simple way avoided deactivation of

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the fragile enzymes and achieved rapid exchange between the inner and outer regions via a semipermeable vesicle wall. In vivo and ex vivo fluorescence verified

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specifically enzyme delivery to the tumor sites due to the EPR effect associated with nanoscale. Significantly, the activity of the enzyme was maintained for a long time (over 4 days).

3. Nanodrugs based on self-assembly of peptide-drug conjugates Rationally designed hydrophobic drugs can be conjugated to hydrophilic elements to form peptide-drug conjugates. The resultant amphiphilic prodrug design focused on the introduction of additional driving forces that can contribute directional interactions to promote new self-assembling features, which opens an avenue for

ACCEPTED MANUSCRIPT optimization of structure and function [69, 90]. Phototherapy, mainly including photodynamic therapy and photothermal therapy, as a minimally invasive, highly safe and precisely targeting modality has been attracting tremendous attention. Light-absorbers as indispensable elements play a significant role in the final dosage forms, which demand specific chromophore arrangement to

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maximize the therapeutic index. The distance between the chromophores can be tailored by peptide-chromophore conjugates because of the fixed stoichiometry and

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partly restricted spatial arrangement [24, 91-93]. More recently, our groups [68]

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synthesized a peptide-porphyrin conjugate (TPP-G-FF). Strong hydrophobic effects of the peptide-porphyrin conjugates motivate its self-assembly into photothermally

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active peptide-porphyrin nanodots (PPP-NDs) with a diameter of 25 ± 10 nm (Figure 3a,b). Narrowing the intermolecular distance led to complete fluorescence quenching

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and inhibition of generating reactive oxygen species, thereby absorbed light energy was transformed into heat to PPP-NDs with high photothermal conversion efficiency

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(54.2%). Most intriguingly, the excellent stability resulting from the hydrophobic

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effects provides a prerequisite for further application, either in vitro or in vivo level. In vivo acoustic imaging verified the tumor site accumulation and retention of PPP-NDs because of preferable passive targeting and long circulation properties

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(Figure 3c). IR thermal imaging demonstrated that the mean temperature at the tumor sites increased to 58.1 °C, much higher than pure irradiation without PPP-ND groups

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under the same conditions (Figure 3d,e). Tumor growth study demonstrated that no tumor recurrence was observed for mice treated with both PPP-NDs and irradiation. In contrast, mice treated with only PPP-NDs or irradiation showed continued and rapid tumor growth, displaying no significant difference with untreated mice. Moreover, the body weights of mice and Hematoxylin and eosin (H&E) sections of the organs suggested, that the PTT treatment by PPP-NDs is biosafe and robust for tumor ablation.

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Fig. 3. (a) Schematic illustration of peptide-porphyrin conjugate (TPP-G-FF) self-assembly into photothermal nanodots (PPP-NDs). (b) TEM image of PPP-NDs. c) PA images of mice at various time points after intravenous

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injection of PPP-NDs. (d) IR thermal images of intravenous PPP-NDs injected mice under continuous irradiation. (c) Mean temperature of the tumor sites as a function of irradiation time.

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Wang and co-workers [92] also developed peptide-photosensitizer conjugates for PTT therapy. Interestingly, the peptide Arg-Gly-Asp (RGD) and photosensitizer

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Purpurin 18 (P18) linked by Pro-Leu-Gly-Val-Arg-Gly (PLGVRG) selectively responds to gelatinase, an enzyme overexpressed in the tumor microenvironment. The

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formed P18-PLGVRGRGD is soluble in physiological conditions and specifically

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binds to the tumor site. In the tumor microenvironment gelatinase cut the responsive linker resulting in hydrophobicity enhancement of the molecules and steric hindrance reduction. Finally, the hydrophobic P18 motif self-assembled into nanofibers in-situ

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due to π-π stacking, therefore exhibiting prolonged retention in the tumor site with overexpressed αvβ3 integrin, which directly led to an enhanced PA signal and PTT

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therapeutic efficacy.

Supramolecular hydrogels formed by amphiphilic hydrogelators can self-assemble into filamentous nanostructures that then entangle to create a three-dimensional network. Xu [94-96] and Yang [97-100] and coworkers designed and constructed drug-peptide amphiphilic hydrogelators to achieve supramolecular hydrogels as a drug self-delivery platform. Cui and co-workers [70, 101] created a “drug amphiphile’’ platform in which a hydrophobic drug was conjugated to a short peptide showing a preference for β-sheet formation. Specifically, they conjugated the hydrophobic drug

ACCEPTED MANUSCRIPT camptothecin (CPT) to a β-sheet-forming peptide sequence derived from the Tau protein through the reducible disulfylbutyrate (buSS) linker [70]. The drug content can be precisely controlled by attachment of one, two or four CPT molecules, corresponding to fixed drug loadings of 23%, 31%, and 38%, respectively. The resulting nanostructures can be either nanofibers or nanotubes depending on the

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number of branching points for attaching CPT molecules. Higher drug content induced a morphology transition from nanofilaments to nanotubes ascribed to the

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increasing hydrophobicity and possible π-π associative interactions among CPT intra-

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and inter-molecular units. The stability test suggested that these nanostructures were stable enough to resist dilution to a certain extent due to the increased hydrophobicity

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and π-interaction as well. Importantly, the formation of nanostructures sequestered the CPT drug into the core and thus protected it from the external environment.

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Biodegradable linkers towards the external environment offer stimuli-responsiveness for controlled release of CPT. An in vitro anti-tumor test demonstrated that these drug

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number of cancer cell lines.

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nanostructures release the bioactive form of CPT and showed good efficacy against a

Later, they extended this “drug amphiphile” strategy to construct bulky paclitaxel (PTX) and planar CPT dual drug amphiphiles [101]. The two anticancer drugs of

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interest were stochastically conjugated to a β-sheet forming peptide (Sup35), and under physiologically relevant conditions the dual DA could spontaneously associate

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into supramolecular filaments with a fixed 41% total drug loading (29% PTX and 12% CPT). The conjugate initially forms small, flexible filamentous structures (7-8 nm in width) and finally transforms into long twisted two-filament fibrils (∼14 nm in width). A possible explanation is, that the initial short filaments are a kinetic product of the hydrophobic collapse, further rearrangement of the internal structure allowing for the formation of β-sheet hydrogen bonds, displaying twisted two-filament fibrils as thermodynamically favored. Additionally, the hetero dual drug amphiphiles were found to effectively release the two anticancer agents, exhibiting superior cytotoxicity

ACCEPTED MANUSCRIPT against PTX-resistant cervical cancer cells. Notably, they also linked Tat cell penetrating peptides (CPPs) and anticancer drugs PTX with a covalent bond for exploration as molecular vector, devoted to improve drug loading, solve the water solubility and overcome multidrug resistance. In the burgeoning field of tumor immunotherapy, the use of peptides as vaccine

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design, especially the adjuvant role they played is experiencing enthusiasm owing to their precise chemical definition [102-105]. Many clinical products of adjuvants are

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hindered mostly due to tedious manufacturability, lack of effectiveness and

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unacceptable levels of tolerability or safety concerns [106]. It is possible, that peptides with desired property play a significant role and are thus investigated widely in the

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vaccine design. To some extent, this refers to Q11 (QQKFQFQFEQQ) conjugated with epitopes capable of inducing either humoral or cellular immune responses as a

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potent peptide vaccine [107-111], where the self-assembling Q11 is a promising

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vaccine adjuvant illuminated by mechanistic studies [108].

Fig. 4. (a,b) Schematic and sequences of epitope-bearing self-assembling peptides. The Q11 domain (blue)

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assembles into fibrillar aggregates, displaying the epitope sequence (red) at the end of a flexible spacer (green). Reprinted with permission from National Academy of Sciences [107]. Schematic illustration of protein-bearing self-assembled peptide nanofibers: (c) Chemical structure of pNP-Q11. (d) Schematic of the non-covalent assembly of Q11 and pNP-Q11 into nanofibers, and the subsequent covalent capture of cutinase-GFP. Reprinted with permission from Macmillan Publishers Limited, part of Springer Nature [113].

Collier and co-workers [107, 108, 112] designed a peptide containing a Q11 self-assembling domain in tandem and OVA323-339 (a 17-amino acid peptide from chicken egg ovalbumin) [107], showing multiple antigenic determinants, including known T and B cell epitopes. The peptides containing Q11 undergo self-assembly in salt-containing aqueous environments to form networks of β-sheet-rich nanofibers

ACCEPTED MANUSCRIPT appearing in the C-terminal peptide extension as long, unbranched fibrils linked epitope-bearing displaying on surface (Figure 4a,b). IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund’s adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. Undetectable levels of interferon-gamma, IL-2, and IL-4 in

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cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help. They [108] furthermore

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verified the T cell-dependent antibody response using T cell knockout models, which

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suggested a route for avoiding or ensuring immunogenicity, and studied the role of the adjuvant. A second self-assembling peptide, KFE8, similar to Q11 in assembly

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property and the nanofiber of OVA-KFE8 also elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the

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specific self-assembling peptide sequence but on the self-assembly. However, the epitope conjugated in the design is restricted to short peptide epitopes, they [112]

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further modified the building block with p-nitrophenyl phosphonate (pNP) ligands in

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a co-assembly adoption with Q11 to form nanofibers with a covalent active site-directed capture of cutinase fusion proteins. This is a model antigen with larger, properly folded and biologically active properties. This nanofiber formulation

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represents precise control of the amount of protein antigen and itself acts as adjuvant in the treatment of mice. Moreover, it [113] employs “β-Tail” tags allowing for high

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protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional Q11 peptides (Figure 4c,d). Multiple different β-Tail fusion proteins could be incorporated into peptide nanofibers alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. Similarly, Li and co-workers [114] applied this strategy in tumor therapy. Yang and co-workers have developed an enzyme-catalyzed [115] method, a heating-cooling process or simply an autoclave assisted phosphate buffer saline method [116] to form

ACCEPTED MANUSCRIPT a hydrogel as a self-adjuvant when mixing peptide and antigen. Other amphiphilic blocks [103, 117] with epitope incorporated have been developed in the vaccine design as potent adjuvant. 4. Protein-modulated formation of assembled nanodrugs The self-assembly of proteins into small-scale complexes plays a crucial biological

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role. Albumin is the most abundant protein in plasma and is of importance in design of versatile nanodrugs for drug delivery [118-120] owing to the inherent merits, such

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as natural structure, biocompatibility, versatile tenability, and unique protein structure

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offering the possibility of modification or conjugation of the surface and targeting to tumor cells mediated by binding of albumin to albumin-binding proteins. Inspired by

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the interactions between protein molecules and drugs in vivo, especially hydrophobic interactions between Bovine serum albumin (BSA) and drugs provide a preferable

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association site in a co-delivery way. Our groups [58] co-assembled the photosensitizer Ce6 and BSA via hydrophobic interaction for formation of Ce6-BSA the Ce6-BSA

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nanoparticles in aqueous solution. Further co-assembly of

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nanoparticles with graphene oxide (GO) in aqueous solution yielded Ce6-BSA-GO nanohybrids via hydrophobic interaction and π-π stacking capable of protecting Ce6. Intriguingly, the nanohybrids showed enhanced cellular uptake and in vitro release of

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the photosensitizer, leading to an improved PDT efficacy. Collagen is an abundant protein existing in mammalian tissues and attracts

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tremendous attention in biomedical applications due to the bio-origin. Collagen proteins such as type I collagen have been developed as the building blocks, which can self-assemble to form fibrous hydrogels via noncovalent interactions. However, the relatively poor mechanical property of collagen hydrogels restrains the future in vivo application. Considering the enhancement of the mechanical properties in a facile and friendly route, our group [121] proposed a collagen protein self-assembly process triggered by biomineralization to endow the hydrogels with adjustable mechanical, shear-thinning and self-healing properties. The process involves

ACCEPTED MANUSCRIPT electrostatic interaction between positively charged collagen chains and negatively charged inorganic [AuCl4]− ions. Subsequently, collagen hydroxyproline residues that can function as a reducing agent to reduce HAuCl4 into gold nanoparticles (AuNPs), playing the role of cross-linkers for flexible and tunable control over the mechanical properties. The rheological characterization of the ultimately obtained collagen-based

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hydrogels highlighted the injectable nature relating to shear-thinning and rapid self-healing properties, which inspired us to further entrap a photosensitizer as a

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model drug as injectable agent for antitumor therapy. Intravital fluorescence imaging

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results demonstrated, that mice injected with collagen-based hydrogels show persistent retention and sustained drug release attributed to the hydrogel network

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entrapment prohibiting diffusion. Measurement of the tumor volume with time verified the efficacy of the collagen-based hydrogel. Therefore, the tumor can be

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irradiated for several times, representing our “one injection, multiple-treatment” strategy, which led to highly effective combinatorial therapeutic efficacy against

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tumor cells.

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5. Conclusions and future perspectives In summary, we have highlighted molecular drugs as building units to create supramolecular nanodrugs through cooperative self-assembly associated with

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intermolecular interactions of both peptides and drugs, in which drugs are no longer considered as passive cargos but participate in their own delivery to desired lesions.

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The concern on purely EPR-based tumor uptake gradually involved to develop “on-off”

dosing,

through

endogenous

stimuli

relating

to

pathological

microenvironment or exogenous stimuli in line with the material itself. Examples we emphasized in this review possess the following features: (i) Biologically relevant peptides endow the eventual nanodrugs with increased biocompatibility and biodegradability; (ii) Feasibility of drugs conjugating with peptides allows introduction of directional forces (such as hydrogen bonding) for assembly; (iii) Collection property of assembled nanodrugs, e.g. it is “hydrophobic effect”, resulting

ACCEPTED MANUSCRIPT from hydrophobic drugs and hydrophobic moieties of peptides, that is critically responsive to drive the formation of nanocolloids and eventually stabilize the assembled nanodrugs. This promotes the circulation stability of nanodrugs in physiological environment, which is of great importance for subsequent tumor accumulation.

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Although amazing progress on design, preparation and application of nanodrugs has been made over decades, a number of challenges still remain in the nanodrug

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evolvement. For instance, diversity and multifunctionality enable the choice of an

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appropriate nanodrug as a thorny problem, for which the physicochemical properties of shape, well-defined surface chemistry, and mechanical parameters are all influence

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factors in body circulation. Moreover, the desired lesion, administration route and dosage are different in various treatments and thus needed for the nanodrug

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formulations with on-demand physicochemical properties. Many available nanodrugs have limited chances of reaching the clinic translation due to immature technique for

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scale-up manufacture and biosafe concerns. These issues are critical barriers for

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nanodrug development and clinic translation. The assembled nanodrugs may provide a new alternative for circumventing the above issues. The self-assembly technique, for preparation of assembled nanodrugs, is facile and efficient, with no need of

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tedious procedures. Nevertheless, one has to consider the influence of fluid-shearing and diffusion on the product quality in the process of scale-up engineering. This could

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be main research topic for scale-up of assembled nanodrugs. Coming from a materials perspective, there is still a need to develop rules to assemble drugs of specific structure and also the structure/property relationship. This is not yet done in this new field, but the chances of success are high as we consider assemblies of only few not too complex molecules. Also, the biologically originated and derived building blocks may be critical for improved biosafety, but we have to consider the immunogenicity provoked by biomacromolecules such as proteins. In some sense, therefore, small biomolecules such as amino acids and peptides may be potential. As regards

ACCEPTED MANUSCRIPT collection properties, there is special concern to consider the biological or physiological environment, and there should be standards developed concerning the requirement for successful treatments. Precise control over the assembled structure may arrive at a platform of assembled nanodrugs as a toolbox for developing a modular treatment by combining these drugs in a dedicated formulation. Every

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sickness or tumor is different, so there will be a need for specialized treatment, now called personalized medicine, which means a need for many different formulations

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and even for one tumor there is not the unique treatment.

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* of special interest.

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** of outstanding interest.

Acknowledgements

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We acknowledge financial support from the National Natural Science Foundation of

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China (Project Nos. 21522307, 21473208, and 91434103), the Talent Fund of the

Sciences

of

the

Chinese

NU

Recruitment Program of Global Youth Experts, the Key Research Program of Frontier Academy

of

Sciences

(CAS,

Grant

No.

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QYZDB-SSW-JSC034), and the CAS President’s International Fellowship Initiative (2017DE0004 and 2017VEA0023). Prof. Möhwald is greatly thanked for his

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consistent support.

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Graphical abstract

ACCEPTED MANUSCRIPT Highlights Recent strategies on design and regulation of assembled nanodrugs associated with precise organization of single or multiple non-covalent interactions were reviewed.

anticancer

therapy

including

chemotherapy,

RI

innovative

PT

Applications are highlighted in some newly emerging fields of

SC

photodynamic/photothermal therapy and immunotherapy.

AC

CE

PT E

D

MA

nanodrugs were also addressed.

NU

The challenges and future perspectives of such kinds of assembled